Our overall goal is to determine the mitochondrial mechanisms that influence neurotransmitter release and the impact of these mechanisms across different synapse types. Mitochondria in nerve terminals are well placed to influence neurotransmitter release but their means of influence have resisted clarification. Many facets of mitochondrial function have been directly implicated in synaptic plasticity but due to the interwoven nature of these activities (ATP production; Ca2+ and Na+ handling; extrusion of protons; release of reactive oxygen species) it has been difficult to identify those that make the primary impact. A second area that requires clarification is the role of mitochondria in different forms of short-term synaptic plasticity. Although mitochondria have an established role in the post-tetanic potentiation of synaptic strength, little is known about their impact on other forms of short-term synaptic plasticity. Lastly, while we know that mitochondria influence neurotransmitter release and synaptic plasticity in large nerve terminals very little is known about their influence in small terminals, typical of the mammalian CNS. These are glaring gaps in our knowledge, particularly as synaptic plasticity allows for changes in synaptic strength, a phenomenon underlying learning and memory. More troubling perhaps, is that mitochondrial dysfunction is found at the epicenter of many neurodegenerative conditions for which the pathogenesis and progression are poorly understood. The central hypothesis is that mitochondria influence neurotransmitter release through multiple mechanisms, and the architecture of the nerve terminal and its firing history determines which mechanism is influential. We bring a combined electrophysiological, imaging and genetic approach to address this hypothesis at Drosophila nerve terminals in vivo, and we introduce a novel peripheral synapse with a single release-site as a model for central synapses with the same architecture. We will test the ability of mitochondrial Ca2+ uptake to limit the amplitude of Ca2+ transients and neurotransmitter release during short trains of action potentials - a firing pattern common in central neurons (Aim 1). Emphasis will be placed on single release-site nerve terminals where we observe mitochondria to have a voracious appetite for Ca2+. We will determine if mitochondria in these terminals are more effective at taking up Ca2+ because they are able to take up Ca2+ directly from Ca2+ microdomains (Aim 2). We will determine whether mitochondrial ATP production, rather than Ca2+ uptake, is the principle mechanism that maintains synchronous release during sustained nerve firing (Aim 3). Finally we will test the requirement for mitochondrial Ca2+ release in the post-tetanic potentiation of transmitter release, and examine the transfer of Ca2+ between mitochondria and the endoplasmic reticulum (Aim 4). An understanding of how mitochondrial function influences synaptic transmission under non-pathological conditions will provide the foundation required to understand the role of mitochondria in pathological conditions.